Veterinary World, EISSN: 2231-0916 Available at www.veterinaryworld.org/Vol.6/Nov-2013/5.pdf RESEARCH ARTICLE Open Access

Background: Infectious bronchitis virus (IBV) affects the upper respiratory tract and the reproductive tract, and some strains can cause nephritis. Large number of serotypes and genotypes of the virus have been identified and for the most part do not cross-protect. Identifying the genotype or serotype of IBV field strains is empirical for selecting an appropriate candidate to serve as vaccine strain for prevention of infectious bronchitis (IB) disease in chickens. The variant strains of IBV could be circulating among chickens in India and recently nephropathogenic IB was reported. Hence, the present work was aimed to carry out molecular characterization of nephropathogenic IBV isolates obtained from two different geographical locations of south India, involving relatively conserved regions. Materials and Methods: MIBVPCR and NIBVPCR primers were used to amplify partial M and N gene after synthesizing cDNA. Isolates Ind/KA/07/1 and Ind/TN/07/2 were subjected to direct sequencing as these two isolates are two different geographical regions and scored better in induction of lesions during pathotyping. Results: Amino acids of membrane glycoprotein varied on five occasions for the isolate Ind/KA/07/1 when aligned with that of M 41 strain and isolate Ind/TN/07/2. Eight amino acids of both isolates in 5b protein were different from that of M41 strain. Few point mutations, short deletions and insertions were noticed in the amplified genome, based on the membrane protein nucleotide sequence comparison. Conclusion: Prevalence of IBV strains with few modifications in conserved regions indicated that there was presence of variant IBV in south India.


Introduction
vaccines available for use, whereas countless different types and variants of the virus capable of causing Infectious bronchitis virus (IBV) is a gamma disease can be found throughout the world.In addition, coronavirus that causes a highly contagious disease in some countries only allow vaccination with one or a chickens.The virus can affect the upper respiratory few vaccine types, making control even more challenging tract and the reproductive tract.In addition, some [1].strains of IBV are reported to be associated with In India, infectious bronchitis (IB) is one of the nephritis as well.Different serotypes and genetic types important poultry diseases and only vaccines prepared of the virus have been identified worldwide and the from Massachusetts strain 41 (M41) is being used.The cross-protection elicited between them is very variant strains of IBV that are antigenically different minimum [1].As IBV exists as multiple different types from vaccine strain could be circulating among that do not cross-protect, it is very difficult to control.chickens in India.The Mass (GenBank accession Attenuated live vaccines are used in broilers and number HM179146) and 793B types were the most pullets and killed vaccines are typically used in layers common IBV types reported in India since 1991 [3]. and breeders.Effective control involves identification Around the year 2000, visceral gout and nephritis was of the virus type causing disease followed by vaccination observed in birds less than 2 wk of age and several with an appropriate vaccine against that type [2].
nephropathogenic and novel strains of IBV were Rapid replication, a high mutation rate, and identified in India, despite vaccination [4,5], China [6, genome recombination results in extensive genetic 7, 8], Slovenia [9], Japan [10,11], Cuba [12], Korea diversity and translates into many different types of the [13], Russia [14] and Brazil [15].In Southeast Asia, virus.The mechanism behind the emergence of new appearance of a new variant of nephropathogenic IBV types and variants of the virus is largely unknown.
is also reported [16,17] whereas in Middle East there However, there are only a few different types of IBV was variant IBV causing respiratory symptoms [18,19].Thus, determining the genotype or serotype of field strains is empirical for selecting an appropriate candidate to act as vaccine strain.Sequencing: Isolates Ind/KA/07/1 and Ind/TN/07/2 carried out using TRIsol Reagent (Genei, Bangaluru, were subjected to direct sequencing using MIBVPCR India) following instructions given by manufacturer.
and NIBVPCR primers.For these two isolates, a bulk Synthesis of cDNA: cDNA was synthesized by using PCR (50 µl) was carried out and PCR products in ® ® Revert Aid First Strand Synthesis kit (Fermentas, agarose gel were purified using Purefast gel extraction USA) as per the recommendations of the manufacturer.
kit (Helini Biomolecules, Chennai, India) and used for The reaction mix for cDNA synthesis was carried out as sequencing.Sequencing with MIBVPCR and follows: NIBVPCR primers was done in automated sequencer 11 µl of RNA and 1 µl of specific forward or using AB prism 96 capillary systems by using o reverse primer were heated at 70 C for 5 min and snap-BigDyesTN kit (Applied Biosystems, USA).The chilled on ice for 1 min.Then the following were complete sequence data for each sample was added: 4 µl of 5 X RT-PCR buffer (250 mM of Tris HCl, constructed by adding the complementary sequence 250 mM KCL, 50 mM of DTT), 2 µl of dNTP (10 mM with the actual data of forward sequencing.The each) and 1 µl of RNAse inhibitor (20U).The contents nucleotide sequence reported in this article has been o were mixed gently and kept at 25 C for 5 min.200 U of submitted to GenBank and published (accession MmLV reverse transcriptase (1 µl) was added and numbers JF730308 and JF730309 for 5b protein of the mixed.This reaction mixture was kept in a thermal isolates Ind/KA/07/1 and Ind/TN/07/2 respectively).amino acids of both isolates in 5b protein were different from that of M41 strain.Nucleotide at positions 27, 76, , 170, 197, 216, 225 and 234 varied from that of At the age of one to three weeks, there was M41 strain that led to coding for different amino acids.outbreak of visceral gout in which there was 10 to 20

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The amino acid arginine was predominant for both the per cent mortality.On postmortem, all dead broilers isolates in substituting amino acids leucine, glutamine chicks showed deposition of urates in visceral organs and cysteine which are coded in M41 strain.and kidney was pale and enlarged (Figure -1).The age Discussion of broiler chicken from which samples were collected, was 8 to 15 days and they were not vaccinated with IB Despite vaccination with Massachusetts (M41) vaccine.However, their parent flocks were vaccinated strain of IBV, there was outbreak of visceral gout in against IB.Nine to eleven day-old ECE of SPF origin which there was 10 to 20 per cent mortality during first were inoculated with 200 µl of the samples through month of life in broilers due to nephropathogenic allantoic cavity and incubated for seven to 8 days at strains of IBV.These findings were in accordance with o the reports made by other authors [4,5].37 C in order to see the typical lesions such as embryo Primers MIBVPCR and NIBVPCR were able to dwarfism and curling of toes.Such lesions were anneal to genome of the field isolates indicating there observed in 11 and two from broiler and layer chickens are no genetic modification such as single nucleotide respectively between third and fourth passage levels polymorphism in the annealing regions of these (Figure -2).
primers.In membrane protein amino acid sequence, RT-PCR for allantoic fluid from IBV positive isolate Ind/KA/07/1 differed from M41 strain which samples was carried out for the amplification of M and led to the conclusion that there is prevalence of variant N genes and the 1020 bp product of field isolates and strain in this region.Although it may be ideal to vaccine strain H120 (Figure -3).
sequence the complete genome or entire S1 gene of the Multiple sequence alignment of both nucleotide isolates to assign the genotype, many workers resorted and amino acid sequence for a total of 169 bp pertaining to sequencing of hypervariable regions alone [21,22].to the coding sequence alone in the membrane Genotyping is an excellent tool for epidemiological glycoprotein region of both isolates along with studies and is a convenient, practical tool for typing reference strain M41 are shown in Figure 4. Amino that can be used best as a means of screening to select acid variations from that of M41 are indicated by star potentially important strains [23].For correct without the letter.Amino acids of membrane comparisons, the whole genome or more conserved glycoprotein when aligned with that of M 41 strain and regions are to be compared as any changes in this isolate Ind/TN/07/2 varied on five occasions for the region will clearly bring out the difference [24,25].isolate Ind/KA/07/1.Nucleotide at position 166 for The suggestions made by these authors are considered both isolates varied from that of M41 strain although  for this study.
alignment for membrane glycoprotein (169 bp) with Isolates Ind/TN/07/2 and Ind/KA/07/1 were reference strain M41 revealed variations.The isolate chosen for sequencing owing to their better lesion Ind/KA/07/1 showed more variation with reference score than other isolates in pathotyping trials involving strain M41 in the alignment than Ind/TN/07/2.all the isolates in broiler chickens and also these two Prevalence of IBV strains with few modifications in isolates from two different geographical regions (data conserved regions indicates that there is presence of variant IBV in south India.not shown).Changes in amino acid sequence may influence cellular tropism.The variation in N-

o
Different serotypes thought to be generated by cycler with a cyclic condition programme of 25 C for o o nucleotide point mutations, insertions, deletions or 10 min, 42 C for 60 min and 70 C for 10 min.The recombination of S1 and other genes could be synthesized cDNA was used either immediately for o circulating among chickens in India.Hence, the present PCR or stored at -20 C. work was aimed at the molecular characterization of Primers for amplification: Previously published nephropathogenic IBV isolates obtained from two primers for PCR amplification of partial M and Ngenes different geographical locations of south India, of IBV [20] were utilized and the sequences are as involving relatively conserved regions.follows; Materials and Methods Forward: 5'-TAA GCT TTC AGT GGC TTG CTA AGT GTG AAC C-3' (MIBVPCR) and Reverse: Virus isolation: On post mortem examination, kidney 5'-TGG ATC CAC CGC TAC CTT CAA ACT TGG showing paleness and enlargement with urate deposits GCG G-3' (NIBVPCR).in the ureter from the commercial broiler chickens reared in south India were collected and transported in Polymerase chain reaction: PCR reaction mix 50% glycerol saline or on ice to the laboratory.The contained 3.0 µl of cDNA, 20 picomoles of samples were subjected for isolation in embryonated MIBVPCR, 20 picomoles of NIBVPCR, 2.5 µl of 10X chicken eggs (ECE) of specific pathogen free (SPF) origin.PCR buffer, 1.0 µl of dNTP (10 mM each), 1.0 µl of Taq DNA polymerase (5 U/ µl) and nuclease free water to Reverse transcriptase PCR (RT-PCR): Allantoic fluid make upto 25 µl.Cycling condition followed was as samples collected from ECE showing IBV-characteristic o follows : initial denaturation 94 C for 3 min; 30 cycles embryo lesions were subjected for amplification of o o o of 94 C for 1 min; 37 C for 2 min; 74 C for 5 min, partial M and N genes of IBV.o followed by final extension of 74 C for 10 min.Viral RNA extraction: Viral RNA extraction was

Figure- 2 .
Figure-2.Embryo showing dwarfism and curling of toes inoculated with kidney specimens of broiler chickens after three passages.Control is on left.

Figure- 3 .
Figure-3.Agarose gel (1.5 per cent) electrophoresis of RT-PCR product obtained by amplifying part of M and N gene by using the primers MIBVPCR and NIBVPCR of field isolates and vaccine strain H120; Lane M: 500 bp marker; Lane 1 to 6: Field isolates; Lane 7: Vaccine strain H120

Figure- 4 .
Figure-4.Multiple sequence alignment of both nucleotides and amino acids (in single letter code) for a total of 169 bp of M gene of isolates Ind/KA/07/1 (Sample A) and Ind/TN/07/2 (Sample B) along with that of M 41 strain.

Figure- 5 .
Figure-5.Multiple sequence alignment of both nucleotides and amino acids (in single letter code) for a total of 249 bp of 5b protein coding gene of isolates Ind/KA/07/1 (Sample A) and Ind/TN/07/2 (Sample B) along with that of M 41 strain.